Heparin and heparan sulfate (HS) are sulfated linear polysaccharides composed of alternating α1-4-linked D-glucosamine (GlcNH2) residues and 1-4 linked uronic acid (α-linkage for L-iduronic acid, IdoA, and β-linkage for D-glucuronic acid, GlcA). Possible modifications are 2-O-sulfation on the uronic acid residues and one or more modifications on the glucosamine residues including N-sulfation, N-acetylation, 6-O-sulfation, and 3-O-sulfation. Heparin is a mixture of polysaccharides that can be considered as special forms of HS with higher levels of sulfation and iduronic acid content per disaccharide repeat unit. Heparin is mostly produced by mast cells and heparan sulfates are produced by different cell types in animals. They are attractive synthetic targets due to their structural complexity which possesses great synthetic challenges and their important roles in regulating cancer growth, blood coagulation, inflammation, assisting viral and bacterial infections, signal transduction, lipid metabolism, and cell differentiation.
Currently, more than a hundred heparan sulfate binding proteins have been identified, and the structure-activity relationship studies (SAS) have revealed the interaction pattern between heparan sulfate and protein, and further directed toward discovering and designing HS mimics. Heparin pentasaccharide sequence H5 (also call DEFGH) GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S is essential for antithrombin III binding and thrombin inhibition activities. Based on the DEFGH structure, a new potential antithrombotic, idraparinux, was synthesized by replacing N-sulfate groups in all three glucosamine residues of heparin pentasaccharide DEFGH with O-sulfates and introducing methyl ethers at the available free hydroxyl groups and showed better anticoagulation activity and longer duration of action than DEFGH. Another pentasaccharide sequence HexA-GlcNS-HexA-GlcNS-IdoA2S has high affinity selectively for FGF-2 (fibroblast grow factor 2), while trisaccharide motif IdoA2S-GlcNS6S-IdoA2S is specific for FGF-1. N-, 2-O- and 6-O-sulfations of the glucosamine residues in HS have been shown to be required for FGF4 binding. Additionally, it has been suggested that the N-acetylated glucosamine region rich in GlcA residues displays structural plasticity and hence could mediate protein interactions. However, the detailed information about sequence requirement of HS that interact with many other proteins is currently unclear due to the lack of the technology of preparing a wide range of structurally defined HS.
Current chemical and enzymatic synthetic methods do not provide convenient access to all possible heparin and HS oligosaccharide sequences. Chemical synthetic approaches are time-consuming and tedious. The production yields decrease dramatically with the increase of the length of the target molecules. Obtaining defined structures longer than octasaccharides remains as a major challenge for chemical synthesis. HS-modifying enzymes have been used with other enzymes to prepare heparin polysaccharides and oligosaccharides with a limited range of sulfation patterns. Due to the complex nature of HS-modifying enzymes, these types of methods do not allow the synthesis of a wide range of HS structures.
Sialic acid-containing oligo- and poly-saccharides belong to another group of sugars implicated in various biological and pathological processes. Sialyltransferases are the key enzymes that catalyze the transfer of a sialic acid residue from cytidine 5′-monophosphate-sialic acid (CMP-sialic acid) to an acceptor to form sialic acid-containing products. They function in processes including cell-cell recognition, cell growth and differentiation, cancer metastasis, immunological regulation, as well as bacterial and viral infection. Besides being prevalent in mammals, sialyltransferases have been found in some pathogenic bacteria. They are mainly involved in the formation of sialic acid-containing capsular polysaccharides (CPS) and lipooligo(poly)saccharides (LOS/LPS), serving as virulence factors, preventing recognition by host's immune system, and modulating interactions with the environment.
Cloning of sialyltransferases from various sources, including mammalian tissues, bacteria, and viruses has been reported. However, most mammalian glycosyltransferases including sialyltransferases—suffer from restricted substrate specificity and no or low expression in laboratory E. coli systems. In comparison, bacterial glycosyltransferases have more promiscuous substrate flexibility and are generally easier to access using E. coli expression systems.
What is needed is a convenient route to form complex oligosaccharide products from simple starting materials. Methods for conversion of monosaccharides and monosaccharide derivatives to chemically and biologically important products, including those containing post-glycosylational modifications and sialic acid moieties, are needed. Importantly, the intermediates and products should be formed in a highly regio- and stereo-selective manner. The one-pot enzymatic methods of the present invention meet this and other needs.